Potassium (K) channels are integral membrane proteins of great molecular and functional diversity, present in practically all mammalian cells. These channels are primarily responsible for maintaining a resting membrane potential and are rapidly activated in response to an external depolarizing stimulus, binding of certain ligands, or changes in the intracellular concentration of calcium or ATP. In the excitable cells such as neurons or cardiac myocytes, K-channels determine the duration of the action potential, thus performing a vital function in the central nervous system and the cardiac functions (reviewed in Rudy, B., Neuroscience 25:729-749, (1988); Halliwell, J. V., in Cook, N. S. (ed.), Potassium Channels: Structure, Classification, Function and Therapeutic Potential, Ellis Horwood Ltd., 348-372, (1990)). The calcium-activated K-channel sub-family consists of at least three discernible ionic currents: a large (BK), an intermediate (IK) and a small (SK) conductive channels (reviewed in Castle, M. A., et al., TINS, 12:59-65, (1989); Haylett, B. G. and D. H. Jenkenson, in Cook, N. S. (ed.), Potassium Channels: Structure, Classification, Function and Therapeutic Potential, Ellis Horwood Ltd., 70-95, (1990); Latorre, R., et al., Ann. Rev. Physiol. 51:385-399, (1989)). These K-channels are activated in response to a rise in the intracellular concentration of calcium [Ca.sup.2+ ]i. In addition to calcium [Ca.sup.2+ ]i, the BK and IK channels are also sensitive to the changes in the membrane potential, whereas SK-channel has no significant voltage sensitivity.
Functionally, the SK-channel is involved in the after hyperpolarization that follows action potentials in many neurons. These include the sympathetic ganglionic neurons, hippocampal neurons, neurosecretory neurons and spinal motor neurons, as well as the skeletal muscle cells (Rudy, B., Neuroscience, 25:729-749, (1988); Latorre, R., et al., Annu. Rev. Physiol. 51:385-399, (1989); Pennefather, P. et al., Proc. Nat'l. Acad. Sci. USA 82:3040-3044, (1985); Marty, A., TINS 12:420-424 (1989); Lancaster, B., et al., Neurosci. 11:23-30 (1991); and Strong, P. N., Pharmac. Ther. 46:137-162, (1990)). Furthermore, the SK-channel has been suggested to play a major role in the spontaneous, transient outward currents in the tracheal smooth muscle cells (Saunders, H. H., et al., J. Pharmacol. Exp. Ther. 257:1114-1119, (1991)), the inhibitory action of the .varies..sub.1 -adrenoceptors, neurotensin receptor and the P2 of the ATP receptor (Haylett, B. G., et al., in Cook, N. S. (ed), Potassium Channels: Structure, Classification, Function and Therapeutic Potantial, 70-95, (1990) and Strong, P. N., Pharmac. Ther., 46:137-162, (1990)).
The neuronal and the skeletal muscle SK-channel is specifically and avidly blocked by a bee venom-derived peptide toxin, apamin (Latorre, R., et al., Annu. Rev. Physiol. 51:385-399, (1989); Moczydlowski, E., et al., J. Membrane Biol. 105:95-111 (1988); Blatz, A. L., et al., J. Gen. Physiol. 84:1-23 (1984); Blatz, A. L. et al., Nature 323:718-720 (1986); and Blatz, A. L., et al., TINS 10:463-467 (1987)). By all indications, the apamin receptor complex is either identical to, or closely associated with, the SK-channel. Apamin is an 18 amino acid neurotoxic peptide which has a single class of binding sites in the rat brain synaptosomes and rat brain slices with an apparent dissociation constant (K.sub.d) of 10-25 pM (Habermann, E., et al., Eur. J. Biochem. 94:355-364 (1979); and Mourre, C., et al., Brain Res. 382:239-249 (1986)). Apamin is also capable of a temperature dependent and high affinity (K.sub.d =30-150 pM) binding to the detergent solubilized brain receptor sites (Seagar, J. J., et al., Biochemistry 25:4051-4057 (1986); Seagar, M. J., et al., Neurosci. 7:565-570 (1987); Schmid-Antomarchi, H., et al., Eur. J. Biochem. 142:1-6 (1984); and Wu, K., et al., Brain Res. 360:183-194 (1985)). The reported B.sub.max value for the rat brain synaptosomes and brain slices is 10-30 fmol/mg protein (Mourre, C., et al., Brain Res. 382:239-249 (1986); Seagar, J. J., et al., Biochemistry 25:4051-4057 (1986); and Wu, K., et al., Brain Res. 360:183-194 (1985)), while that for the detergent solubilized receptor ranges from 0.45 to 17 fmol/mg protein (Seagar, M. J., et al., Neurosci. 7:565-570 (1987); and Schmid-Antomarchi, H., et al., Eur. J. Biochem. 142:1-6 (1984)).
The polypeptide components of the apamin receptor have been studied by several groups. Crosslinking experiments using [.sup.125 ]apamin, followed by SDS-PAGE and autoradiography have indicated that the apamin binding proteins of the rat brain synaptosomal membrane consists of two protein species, a major 80-86 KDa protein and, in most reported preparations, a minor 50-59 KDa band (Seagar, J. J., et al., Biochemistry 25:4051-4057 (1986); Seagar, M. J., et al., J. Biol. Chem. 260:3895-3898 (1985); and Leveque, C., et al., FEBS Letters 275:185-189 (1990)). Partial peptide mapping of the two protein bands using an anti-apamin anti-serum has shown that the smaller polypeptide is likely to be a proteolytic fragment of the larger protein and not an additional subunit of the apamin binding protein in the brain. Furthermore, in the plasma membrane of the cultured neurons or astrocytes, there are additional components with the ability to crosslink to [.sup.125 I]apamin. Crosslinking of [.sup.125 I]apamin to the membranes from the rat heart, liver and smooth muscle has also indicated that an 85-87 KDa polypeptide is the major labeled component of the apamin binding complex (Marqueze, B., et al., Biochem 169:295-298 (1987)). A second 59 KDa protein was identified in the liver membrane only (Marqueze, B., et al., Biochem 169:295-298 (1987)).
The blocking of the small conductance calcium activated potassium channel (sKca) results in prolongation of the action potential, while its activation by an increase in the intracellular calcium concentration accelerates the rate of hyperpolarization, thus shortening the duration of the action potential. In vascular smooth muscle cells (such as those in veins and arteries), activation of sKca results in the hyperpolarization of the smooth muscle membrane, which in turn results in the inhibition of the voltage-gated calcium channels. The inhibition of the latter may then lead to the relaxation of the blood vessels and lowering of the blood pressure. In the heart, modulation of sKca can be a potentially useful means to regulate an arrhythmic heart. In the nervous system, the hypocampus of Alzheimer's patients shows a drastic reduction in apamin density (Vaitukatis, J. L., et al., Methods in Enzymology 73:46-52 (1981)). Further, apamin receptor in neurons has been reported to be involved in the process of learning and memory (Messier, C., et al., Brain Res. 551:322-326 (1991)). Thus, manipulation of this receptor may also result in improving cognition. Notwithstanding the significant therapeutic potential manipulation of sKca may have, relatively little is known about the identity of the proteins involved in this channel. The present invention now provides key elements in the study of the potassium channel function.